The cloned, functional mouse pituitary adenocarcinoma cell line, AtT-20, is grown in large-volume suspension culture. Using bioassays for ACTH and MSH and radioimmunoassays for ACTH, alpha-MSH, and beta- MSH, it has been shown that this single cell line synthesizes and secretes both ACTH and MSH, which can be physically separated by gel filtration on Sephadex G-50 fine resin. Synthesis of ACTH is specifically inhibited by glucocorticoids, but not by other steroid hormones. Inhibition is detectable with 10 to the minus 9th power M dexamethasone. A cytoplasmic macromolecular steroid receptor has been identified whose binding affinity for various steroids is proportional to their inhibitory effects on ACTH synthesis. The dissociation constant with H3-triamcinolone acetonide was 2.3 times 10 to the minus 9th power M. The receptor-steroid complex has a sedimentation coefficient of 4S in high salt. The receptor appears to be a protein. The AtT-20 cell also produces a C-type particle which appears to be a Moloney murine leukemia virus by electron microscopy, indirect immunofluorescence, and XC syncytial cell formation assay. Human cell lines, derived from an amelanotic malignant melanoma causing the ectopic ACTH syndrome, have been established in tissue culture and cloned. They continue to synthesize and secrete ACTH after two years in continuous culture.